Estrogens are used in medicine for hormone replacement therapy. In particular, estrogen mixtures are used for the treatment and prophylaxis of the disorders of the climacteric period which occur in women after natural or artificial menopause. The mixtures of estrogens such as those found in the urine of pregnant mares are proved to be particularly effective and readily compatible for treatment of such disorders.
Mixture of estrogens include conjugated estrogens salts comprising estrone, equilin, Δ8,9-dehydroestrone, 17α-estradiol; 17α-dihydroequilin, 17α-dihydroequilin, 17α-estradiol, equilenin, 17α-dihydroequilenin; 17β-dihydroequilenin and optionally also one or more conjugated salts from the group of 17β-Δ8,9-dehydroestradiol; 17α-Δ8,9-dehydroestradiol; 6-0H 17α-dihydroequilenin; 6-0H equilenin; 6-0H 17β-dihydroequilenin and/or other sulphated steroidal metabolites. Salts are preferably sodium salts, while conjugated are mainly sulfates.
The amount of estrogens present in the pregnant mare's urine (PMU) may vary largely between the lots. PMU generally contains urea, steroids including estrogens, phenolic constituents which include cresols and dihydro-3,4-bis[(3hydroxyphenyl) methyl]-2(3H)-furanone (HPMF) and other urinary impurities.
The U.S. Pat. No. 2,551,205; U.S. Pat. No. 2,696,265; U.S. Pat. No. 2,711,988 and U.S. Pat. No. 2,834,712 disclose process for the isolation of a mixture of estrogens from pregnant mare's urine by liquid-liquid extraction method. Such liquid-liquid extraction methods have accompanying problems such as severe foaming, sedimentation, emulsification and poor phase separation, which generates large amounts of hazardous waste and therefore are difficult to practice at the commercial scale.
U.S. Pat. No. 5,814,624 discloses a method for obtaining a mixture of conjugated estrogens from pregnant mare's urine comprising adsorption of mixture of estrogens from PMU on a resin and eluting estrogens-loaded resin with an aqueous buffer solution.
U.S. Pat. No. 7,439,235 discloses a method for obtaining a mixture of conjugated estrogens, depleted in phenolic constituents, from pregnant mare's urine comprising adsorption of mixture of conjugated estrogens from PMU on a resin and eluting conjugated estrogens loaded resin with aqueous alkaline solution. The aqueous alkaline solution eluate is expected to remove the phenolic constituents and the mixture of conjugated estrogens was isolated from the fractions containing the desired amount of the product.
U.S. publication No. 2009/0312293 A1 discloses a similar process wherein the resin used for adsorption of mixture of conjugated estrogens from PMU is porous styrene-divinyl benzene polymer brominated at the styrene and/or divinylbenzene portion, with 600 m2/g area, 1.3 ml/g volume (dry weight), about 200 Angstrom pore size and eluting the estrogens from the resin with water: water-miscible solvent.
Canadian patent CA 2263757 discloses a method for isolation of a mixture of conjugated estrogens, depleted in phenolic urine contents, from pregnant mare's urine by using water miscible organic solvent or a mixture of water miscible organic solvent and water which has optionally been rendered alkaline.
The reported procedures involve usage of water alone or in combination with other solvents optionally in alkaline condition as an elution liquid for the purpose of purification and/or isolation of estrogens from the resin loaded with estrogens.
U.S. Pat. No. 8,349,819 discloses a method for purification and isolation of a mixture of estrogens involving the usage of multiple resins, which makes the process less economic.
There remains a need to provide an improved purification and isolation method for obtaining a mixture of estrogens from pregnant mare's urine which is simple, economic and industrially viable, which avoids usage of multiple resins and tedious isolation methods.